The overall aim of this project is to initiate and validate novel methods to discover the genetic program that underlies genitourinary smooth muscle development, using bladder smooth muscle as a model. This tissue has been selected because it is a rich source of smooth muscle cells and also makes use of existing expertise in our respective laboratories. To approach these issues, we first propose to generate transgenically modified mice in which the smooth muscle cells of the genitourinary tract will be "tagged" with Enhanced Green Fluorescent Protein (EGFP) or Enhanced Yellow Fluorescent Protein (EYFP). This will provide an efficient means to identify and isolate smooth muscle cells from these tissues in subsequent experiments. Using these transgenic mice, we will apply Fluorescent Activated Cell Sorting (FACS) methods to isolate purified populati6ns of "immature" and "mature" smooth muscle cells from fetal, newborn and adult bladders. Ultimately, we hope to extend these analyses to the earliest smooth muscle-related cells in the urogenital sinus. Lastly, we will use microchip arrays and suppressive subtractive hybridization (SSH) strategies applied to the purified "tagged" cells to identify those genes, which are involved in the differentiation of bladder smooth muscle cells. This R21 proposal is expected to define the expression of known and novel genes whose mRNA expression varies during the development of the bladder smooth muscle of FVB/N mice using microarray technology. We believe that these studies will yield valuable insights into alterations in genetic programs that are involved in bladder development. This will provide baseline data for future studies which can be extended to other genitourinary tissues and organs (including the prostate) to approach issues relating to hypertrophy, involution, injury response and recovery, and cancer. We also expect to generate a number of reagents (transgenic animals with fluorescently labeled smooth muscle and custom genitourinary DNA chips) that will prove of value to the scientific community.